PROJECT SUMMARY/ABSTRACT Our overarching objective is to define key DNA repair, replication and cell cycle mechanisms that support accurate chromosome transmission. This is highly significant because the faithful propagation of chromosomes suppresses human disease, including cancer and aneuploidy-related birth defects. Our proposal centers on the conserved Smc5-Smc6 ?Structural Maintenance of Chromosomes (SMC)? complex which, as a linchpin of genome stability and DNA repair, suppresses aging phenotypes and tumorigenesis. Using biochemical, genetic and mass spectrometry approaches in the proven fission yeast model organism, we defined the subunit composition and architecture of the octameric Smc5-Smc6 holocomplex. Since then, mainly genetic approaches have revealed critical roles for Smc5-Smc6 in DNA repair via homologous recombination, and chromosome segregation during both mitosis and meiosis. However, a mechanistic understanding of Smc5- Smc6 function(s) in these processes is lacking, a key knowledge gap that we are ideally suited to address through our three focused and integrated Specific Aims. (1) Smc5-Smc6 loads non-randomly across the genome, and upon genotoxic stress it becomes enriched at certain loci to protect genetic integrity. However, how this dynamic Smc5-Smc6 chromatin association is controlled remains largely undefined. Therefore, using chromatin immunoprecipitation methods, coupled to deep sequencing and/or quantitative PCR, we will determine how key Smc5-Smc6 associated factors affect its genome-wide chromatin association. (2) SMC complexes share the ability to topologically entrap DNA within their ring-like structures, a property that is central to their roles in chromosome segregation and repair. However, mechanisms and cofactors that promote Smc5-Smc6 topological loading on DNA remain undefined. Therefore, we will use our purified Smc5-Smc6 complexes in a biochemically defined in vitro assay to test the role(s) of candidate Smc5-Smc6 DNA loading cofactors. In addition, we will test if DNA binding exhibited by the candidate Smc5-Smc6 loading cofactors is structure selective, which could promote Smc5-Smc6 loading at certain DNA lesions in vivo. (3) We revealed a critical role for Smc5-Smc6 in the processing of covalent linkages between chromosomes called Holliday junctions (HJs) which, to suppress genetic instability and disease, must be removed before chromosomes attempt to segregate in mitosis and meiosis. Notably, through as yet undefined mechanisms, Smc5-Smc6 promotes HJ removal by an endonuclease we discovered called Mus81-Eme1. To determine how Smc5-Smc6 regulates Mus81-Eme1, we will define the proteomic environment of Smc5-Smc6 during HJ processing using a novel approach of genetic activation coupled to proximity-based biotinylation and protein identification methods. Overall, it is clear that Smc5-Smc6 is a fundamental mediator of genetic integrity, and revealing the mechanisms and breadth of Smc5-Smc6's impact in human disease is the overarching goal of this project.